Aminoglycosides have a significant role in the antimicrobial therapy, especially for the treatment of Gram negative infections. Despite their well-known toxicity, aminoglycosides are preferred because they are suitable for the combination of the antibacterial therapy, and they have a good antibacterial efficiency and a limited tendency toward of the development of resistance during therapy. The concentration-dependent bactericidal activity and prolonged post-antibiotic effect of aminoglycosides have provided that they are widely used for antibacterial therapy. Isepamicin which is previously known SCH 21420 or 1-N-HAPAGentamicin B is a novel broad-spectrum aminoglycoside. In this study, antibacterial susceptibilities of 304 Gram negative bacteria isolated from various clinical specimens against isepamicin, gentamicin, tobramicin, netilmicin, and amikacin were investigated by disk diffusion method in accordance with NCCLS standards. Distribution of 304 strains were 63.8 (n:194) E. coli, 12.5% (n:38) Klebsiella spp., 9.9% (n:30) Pseudomonas spp., 6.6% (n:20), Enterobacter spp., and 7.2 (n:22) Proteus spp., Serratia spp., Citrobacter spp., Salmonella paratyphi Aand Providencia spp., The ratios of antibiotic susceptibility of these bacteria were found as following: for amikacin 87.8%, gentamicin 66.5%, netilmicin 84.9%, tobramycin 65.1%, and isepamicin 94.7%. Thus, the most affective antibiotic to the bacteria was isepamicin, and the least affective one was tobramycin. In conclusion, isepamicin is a suitable alternative antibiotic for the treatment of Gram negative enteric- and non fermentative bacterial-infections.